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MUC1 (D9O8K) XP® Rabbit mAb (PE Conjugate) #19926
Gallery: MUC1 (D9O8K) XP® Rabbit mAb (PE Conjugate) #19926
Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.
NOTE: If live cell staining is desired, proceed to Section C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
- Fix for 15 minutes at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells.
- If necessary, centrifuge to remove excess PBS.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 30-60 minutes at room temperature.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.
D. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted January 2009
revised June 2017
MUC1 (D9O8K) XP® Rabbit mAb (PE Conjugate) detects endogenous levels of total MUC1 protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human MUC1 protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated MUC1 (D9O8K) XP® Rabbit mAb #14161.
Mucins represent a family of glycoproteins characterized by repeat domains and dense O-glycosylation (1). MUC1 (or mucin 1) is aberrantly overexpressed in most human carcinomas. Increased expression of MUC1 in carcinomas reduces cell-cell and cell-ECM interactions. MUC1 is cleaved proteolytically, and the large ectodomain can remain associated with the small 25 kDa carboxy-terminal domain that contains a transmembrane segment and a 72-residue cytoplasmic tail (1). MUC1 interacts with ErbB family receptors and potentiates ERK1/2 activation (2). MUC1 also interacts with β-catenin, which is regulated by GSK-3β, PKCγ, and Src through phosphorylation at Ser44, Thr41, and Tyr46 of the MUC1 cytoplasmic tail (3-5). Overexpression of MUC1 potentiates transformation (6) and attenuates stress-induced apoptosis through the Akt or p53 pathways (7,8).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.